The invention relates to the psoriastatin type I and II genes, psoriastatin type I; and II polypeptides, and methods of using psoriastatin nucleic acids and polypeptides.
Psoriasis is a disease chacterized by abnormal proliferation of keratinocytes and inflammation of the involved skin. A variety of proteinases which are not found in normal epidermis has been reported to appear in such skin.
In general, the invention features a psoriastatin type I polypeptide, e.g., a polypeptide, the sequence of which includes, or is, all or part of the sequence shown in SEQ ID NO:2. Preferred embodiments include fragments and analogs of SEQ ID NO:2, preferably having at least one biological activity of a psoriastatin type I polypeptide.
In preferred embodiments, the polypeptide is a recombinant or a substantially pure preparation of a psoriastatin type I polypeptide.
In preferred embodiments, the residue at position 357 (according to the numbering system used herein) is: other than alanine; a residue having a side chain group of greater molecular weight from the side chain group of alanine; a basic residue; an acidic residue; a threonine.
In preferred embodiments: the polypeptide has at least one biological activity, e.g., it reacts with an antibody, or antibody fragment, specific for a psoriastatin type I polypeptide; the polypeptide includes an amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:2; the polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:2; the polypeptide is at least 5, 10, 20, 50, 100, 150, 200, or 250 amino acids in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, 150, 200, or 250 contiguous amino acids from SEQ ID NO:2; the polypeptide is preferably at least 10, but no more than 100, amino acids in length; the psoriastatin type I polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring psoriastatin type I polypeptide.
In preferred embodiments: the psoriastatin type I polypeptide is encoded by the nucleic acid sequence of SEQ ID NO:1, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with the nucleic acid of SEQ ID NO:1. For example, the psoriastatin type I polypeptide can be encoded by a nucleic acid sequence which differs from a nucleic acid sequence of SEQ ID NO:1 due to degeneracy in the genetic code.
In a preferred embodiment the psoriastatin type I polypeptide is an agonist of a naturally-occurring mutant or wild type psoriastatin type I polypeptide (e.g., a polypeptide having an amino acid sequence shown in SEQ ID NO:2). In another preferred embodiment, the polypeptide is an antagonist which, for example, inhibits an undesired activity of a naturally-occurring psoriastatin type I polypeptide (e.g., a mutant polypeptide).
In a preferred embodiment, the psoriastatin type I polypeptide differs in amino-acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:2. The differences, however, are such that the psoriastatin type I polypeptide exhibits at least one biological activity of a psoriastatin type I polypeptide, e.g., the psoriastatin type I polypeptide retains a biological activity of a naturally occurring psoriastatin type I polypeptide.
In preferred embodiment the psoriastatin type I polypeptide includes a psoriastatin type I polypeptide sequence, as described herein, as well as other N-terminal and/or C-terminal amino acid sequences.
In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:2, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:2.
In yet other preferred embodiments, the psoriastatin type I polypeptide is a recombinant fusion protein having a first psoriastatin type I polypeptide portion and a second polypeptide portion having an amino acid sequence unrelated to a psoriastatin type I polypeptide. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In a preferred embodiment, the psoriastatin type I polypeptide is a fragment or analog of a naturally occurring psoriastatin type I polypeptide which inhibits reactivity with antibodies, or F(abxe2x80x2)2 fragments, specific for a naturally occurring psoriastatin type I polypeptide.
In a preferred embodiment, the psoriastatin type I polypeptide includes a sequence which is not present in the mature protein.
In a preferred embodiment, the psoriastatin type I polypeptide has a molecular weight of about 43 kDa.
In preferred embodiments, the psoriastatin type I polypeptide has one or more of the following properties: the residue at position 357 is other than alanine: has a molecular weight of about 43 kDa; can be isolated from psoriatic tissue; is expressed in psoriatic tissue. e.g., psoriatic epidermis, at least 2, and preferably at least 5 or 10 times more abundantly than in normal tissue; is a cross-class inhibitor (e.g., it inhibits cysteine proteinases, e.g., cathepsin L. but does not inhibit serine proteinases); inhibits cathepsin L at least 2. more preferably at least 2, 5, 10, or 100 times less efficiently than does squamous cell carcinoma-antigen (SCC-A); inhibits cathepsin L but not cathepsin B or cathepsin H; is active at pH 5.0; is secreted.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
The invention includes an immunogen which includes an active or inactive psoriastatin type I polypeptide, or an analog or a fragment thereof, in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the psoriastatin type I polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:2.
The invention also includes an antibody preparation, preferably a monoclonal antibody preparation, specifically reactive with an epitope of the psoriastatin type I immunogen or generally of a psoriastatin type I polypeptide.
In another aspect, the invention provides a substantially pure nucleic acid having, or comprising, a nucleotide sequence which encodes a polypeptide, the amino acid sequence of which includes, or is, the sequence of a psoriastatin type I polypeptide, or analog or fragment thereof.
In preferred embodiments, the nucleic acid encodes a polypeptide having one or more of the following characteristics: at least one biological activity of a psoriastatn type I polypeptide, e.g., a polypeptide specifically reactive with an antibody, or antibody fragment, directed against a psoriastatin type I polypeptide; an amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:2; an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:2, the polypeptide is at least 5, 10, 20, 50, 100, 150, 200, or 250 amino acids in length; at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, 150, 200, or 250 contiguous amino acids from SEQ ID NO:2; an amino acid sequence which is preferably at least 10, but no more than 100, amino acids in length; the ability to act as an agonist or an antagonist of a biological activity of a naturally occurring psoriastatin type I polypeptide.
In preferred embodiments: the nucleic acid is or includes the nucleotide sequence of SEQ ID NO:1; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence of SEQ ID NO:1; the nucleic acid includes a fragment of SEQ ID NO:1 which is at least 25, 50, 100, 200, 300, 400, 500, or 1,000 bases in length; the nucleic acid differs from the nucleotide sequence of SEQ ID NO:1 due to degeneracy in the genetic code.
In preferred embodiments: the nucleotide residue at position 161 is other than an adenine nucleotide, e.g., it is a guanine nucleotide; the nucleotide residue at position 259 is other than an adenine nucleotide, e.g. it is a guanine nucleotide; the nucleotide residue at position 788 is other than an adenine nucleotide, e.g., it is a guanine nucleotide; the nucleotide residue at position 799 is other than an adenine nucleotide, e.g., it is a guanine nucleotide; the nucleotide residue at position 1008 is other than an adenine nucleotide, e.g., it is a guanine nucleotide; the nucleotide residue at position 1090 is other than a guanine nucleotide, e.g., it is an adenine nucleotide.
In a preferred embodiment the polypeptide encoded by the nucleic acid is an agonist which, for example, is capable of enhancing an activity of a naturally-occurring mutant or wild type psoriastatin type I polypeptide. In another preferred embodiment, the encoded polypeptide is an antagonist which, for example, inhibits an undesired activity of a naturally-occurring psoriastatin type I polypeptide (e.g., a polypeptide having an amino acid sequence shown in SEQ ID NO:2).
In a preferred embodiment, the encoded psoriaststin type I polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:2. The differences, however, are such that the encoded psoriastatin type I polypeptide exhibits at least one biological activity of a naturally occurring psoriastatin type I polypeptide (e.g., the psoriastatin type I polypeptide of SEQ ID NO:2).
In preferred embodiments, the nucleic acid encodes a psoriastatin type I polypeptide which includes a psoriastatin type I polypeptide sequence, as described herein, as well as other N-terminal and/or C-terminal amino acid sequences.
In preferred embodiments, the nucleic acid encodes a polypeptide which includes all or a portion of an amino acid sequence shown in SEQ ID NO:2, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:2.
In preferred embodiments, the encoded polypeptide is a recombinant fusion protein having a first psoriastatin type I polypeptide portion and a second polypeptide portion having an amino acid sequence unrelated to a psoriastatin type I polypeptide. The second polypeptide portion can be, e.g., any of glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain. In preferred embodiments the fusion protein can be used in a two-hybrid assay.
In preferred embodiments, the encoded polypeptide is a fragment or analog of a naturally occurring psoriastatin type I polypeptide which inhibits reactivity with antibodies, or F(abxe2x80x2)2 fragments, specific for a naturally occurring psoriastatin type I polypeptide.
In preferred embodiments, the nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the psoriastatin type I gene sequence.e.g., to render the psoriastatin type I gene sequence suitable for use as an expression vector.
In yet another preferred embodiment, the nucleic acid of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotiles from SEQ ID NO:1. or more preferably to at least 20 consecutive nucleotides from SEQ ID NO:1, or more preferably to at least 40 consecutive nucleotides from SEQ ID NO:1.
In a preferred embodiment, the nucleic acid encodes a mature polypeptide having a molecular weight of about 43 kDa.
In a preferred embodiment, the nucleic acid encodes a psoriastatin type I polypeptide which includes a sequence which is not present in the mature protein.
In preferred embodiments, the nucleic acid encodes a psoriastatin type I polypeptide which has one or more of the following properties: the residue at position 357 is other than alanine; has a molecular weight of about 43 kDa; can be isolated from psoriatic tissue; is expressed in psoriatic tissue, e.g., psoriatic epidermis, at least 2, and preferably at least 5 or 10 times more abundantly than in normal tissue; is a cross-class inhibitor (e.g., it functionally inhibits cysteine proteinases, e.g., cathepsin L, but does not inhibit serine proteinases); inhibits cathepsin L at least 2, more preferably at least 2, 5, 10, or 100 times less. efficiently than does squamous cell carcinoma-antigen (SCC-A); inhibits cathepsin L but not cathepsin B or cathepsin H; is active at pH 5.0; is secreted.
In another aspect, the invention includes: a vector including a nucleic acid which encodes a psoriastatin type I polypeptide, e.g., a psoriastatin type I polypeptide; a host cell transfected with the vector; and a method of producing a recombinant psoriastatin type I like polypeptide, e.g., a psoriastatin type I polypeptide; including culturing the cell, e.g., in a cell culture medium, and isolating the psoriastatin type I-like polypeptide, e.g., a psoriastatin type I polypeptide, e.g., from the cell or from the cell culture medium.
In another aspect, the invention features, a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleotide sequence shown in SEQ ID NO:1.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO:1, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In general, the invention features a psoriastatin type II polypeptide, e.g., a polypeptide, the sequence of which includes, or is, all or part of the sequence shown in SEQ ID NO:4. Preferred embodiments include fragments and analogs of SEQ ID NO:4, preferably having at least one biological activity of a psoriastatin type II polypeptide.
In preferred embodiments, the polypeptide is a recombinant or a substantially pure preparation of a psoriastatin type II polypeptide.
In preferred embodiments: the reactive site of the psoriastatin type II polypeptide, e.g., amino acid residues 351-357 (according to the numbering system used herein) have no homology or are less than about 25%, more preferably less than about 42%, 57%, 71% or 85% homologous with the corresponding residues of SCC-A; residues 351-357 are other than GFGSSPA (SEQ ID NO:5); residues 351-357 are WELSSP (SEQ ID NO:6), or a sequence with at least about 14%, 28%, 42%, 057%, 071% or 85% homology therewith
In preferred embodiments: the polypeptide has at least one biological activity, e.g., it reacts with an antibody, or antibody fragment, specific for a psoriastatin type II polypeptide; the polypeptide includes an amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:4; the polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:4; the polypeptide is at least 5, 10, 20, 50, 100, 150, 200, or 250 amino acids in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, 150, 200, or 250 contiguous amino acids from SEQ ID NO:4; the polypeptide is preferably at least 10, but no more than 100, amino acids in length; the psoriastatin type II polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring psoriastatin type II polypeptide.
In preferred embodiments: the psoriastatin type II polypeptide is encoded by the nucleic acid sequence of SEQ ID NO:3, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with the nucleic acid of SEQ ID NO:3. For example, the psoriastatin type II polypeptide can be encoded by a nucleic acid sequence which differs from a nucleic acid sequence of SEQ ID NO:3 due to degeneracy in the genetic code.
In a preferred embodiment the psoriastatin type II polypeptide is an agonist of a naturally-occurring mutant or wild type psoriastatin type II polypeptide (e.g., a polypeptide having an amino acid sequence shown in SEQ ID NO:4). In another preferred embodiment, the polypeptide is an antagonist which, for example, inhibits an undesired activity of a naturally-occurring psoriastatin type II polypeptide (e.g., a mutant polypeptide).
In a preferred embodiment, the psoriastatin type II polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:4. The differences, however, are such that the psoriastatin type II polypeptide exhibits at least one biological activity of a psoriastatin type II polypeptide, e.g., the psoriastatin type II polypeptide retains a biological activity of a naturally occurring psoriastatin type II polypeptide.
In preferred embodiments the psoriastatin type II polypeptide includes a psoriastatin type II polypeptide sequence, as described herein, as well as other N-terminal and/or C-terminal amino acid sequences.
In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:4, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:4.
In yet other preferred embodiments, the psoriastatin type II polypeptide is a recombinant fusion protein having a first psoriastatin type I polypeptide portion and a second polypeptide portion having an amino acid sequence unrelated to a psoriastatin type II polypeptide. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain In preferred embodiment the fission protein can be used in a two-hybrid assay.
In a preferred embodiment, the psoriastatin type II polypeptide is a fragment or analog of a naturally occurring psoriastatin type II polypeptide which inhibits reactivity with antibodies, or F(abxe2x80x2)2 fragments, specific for a naturally occurring psoriastatin type II polypeptide.
In a preferred embodiment, the psoriastatin type II polypeptide includes a sequence which is not present in the mature protein.
In a preferred embodiment, the psoriastatin type II polypeptide has a molecular weight of about 43 kDa:
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
In preferred embodiments, the psoriastatin type II polypeptide: has a molecular weight of about 43 kDa; can be isolated from psoriatic tissue; is expressed in psoriatic tissue, e.g., psoriatic epidernis, at least 2, and preferably at least 5 or 10 times more abundantly than in normal tissue; is localized in the nucleus of the cell in psoriatic tissue.
The invention includes an immunogen which includes an active or inactive psoriastatin type II polypeptide, or an analog or a fragment thereof, in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the psoriastatin type II polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:4.
The invention also includes an antibody preparation, preferably a monoclonal antibody preparation, specifically reactive with an epitope of the psoriastatin type II immunogen or generally of a psoriastatin type II polypeptide.
In another aspect, the invention provides a substantially pure nucleic acid having, or comprising, a nucleotide sequence which encodes a polypeptide, the amino acid sequence of which includes, or is, the sequence of a psoriastatin type II polypeptide, or analog or fragment thereof.
In preferred embodiments: the nucleic acid encodes a reactive site of the psoriastatin type II polypeptide, e.g., amino acid residues 351-357 (according to the numbering system used herein) have no homology or are less than about 25%, more preferably less than about 42%, 57%, 71% or 85% homologous with the corresponding residues of SCC-A; residues 351-357 are other than GFGSSPA (SEQ ID NO:5); residues 351-357 are VVELSSP (SEQ ID NO:6), or a sequence with at least about 14%, 28%, 42%, 57%, 71% or 85% homology therewith.
In preferred embodiments, the nucleic acid encodes a polypeptide having one or more of the following characteristics: at least one biological activity of a psoriastatin type II polypeptide, e.g., a polypeptide specifically reactive with an antibody, or antibody fragment, directed against a psoriastatin type II polypeptide; an amino acid sequence at least; 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:4; an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:4, the polypeptide is at least 5, 10, 20, 50, 100, 150, 200, or 250 amino acids in length; at: least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, 150, 200, or 250 contiguous amino acids from SEQ ID NO:4; an amino acid sequence which is preferably at least 10, but no more than 100, amino acids in length; the ability to act as an agonist or an antagonist of a biological activity of a naturally occurring psoriastatin type II polypeptide.
In preferred embodiments: the nucleic acid is or includes the nucleotide sequence of SEQ ID NO:3; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence of SEQ ID NO:3; the nucleic acid includes a fragment of SEQ ID NO:3 which is at least 25, 50, 100, 200, 300, 400, 500, or 1,000 bases in length; the nucleic acid differs from the nucleotide sequence of SEQ ID NO:3 due to degeneracy in the genetic code.
In a preferred embodiment the polypeptide encoded by the nucleic acid is an agonist which, for example, is capable of enhancing an activity of a naturally-occurring mutant or wild type psoriastatin type II polypeptide. In another preferred embodiment, the encoded polypeptide is an antagonist which, for example, inhibits an undesired activity of a naturally-occurring psoriastatin type II polypeptide (e.g., a polypeptide having an amino acid sequence shown in SEQ ID NO:4).
In a preferred embodiment, the encoded psoriastatin type II polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:4. The differences, however, are such that the encoded psoriastatin type II polypeptide exhibits at least one biological activity of a naturally occurring psoriastatin type II polypeptide (e.g., the psoriastatin type II polypeptide of SEQ ID NO:4).
In preferred embodiments, the nucleic acid encodes a psoriastatin type II polypeptide which includes a psoriastatin type II polypeptide sequence, as described herein, as well as other N-terminal and/or C-terminal amino acid sequences.
In preferred embodiments, the nucleic acid encodes a polypeptide which includes all or a portion of an amino acid sequence shown in SEQ ID NO:4, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:4.
In preferred embodiments, the encoded polypeptide is a recombinant fusion protein having a first psoriastatin type II polypeptide portion and a second polypeptide portion having an amino acid sequence unrelated to a psoriastatin type II polypeptide. The second polypeptide portion can be, e.g., any of glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain. In preferred embodiments the fusion protein cane be used in a two-hybrid assay.
In preferred embodiments, the encoded polypeptide is a fragment or analog of a naturally occurring psoriastatin type II polypeptide which inhibits reactivity with antibodies, or F(abxe2x80x2)2 fragments, specific for a naturally occurring psoriastatin type II polypeptide.
In preferred embodiments, the nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the psoriastatin type II gene sequence, e.g., to render the psoriastatin type II gene sequence suitable for use as an expression vector.
In yet another preferred embodiment, the nucleic acid of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides from SEQ ID NO:3, or more preferably to at least 20 consecutive nucleotides from SEQ ID NO:3, or more preferably to at least 40 consecutive nucleotides from SEQ ID NO:3.
In a preferred embodiment, the nucleic acid encodes a mature polypeptide having a molecular weight of about 43 kDa.
In a preferred embodiment, the nucleic acid encodes a psoriastatin type II polypeptide which includes a sequence which is not present in the mature protein.
In preferred embodiments, the nucleic acid encodes a psoriastatin type II polypeptide which: has a molecular weight of about 43 kDa; can be isolated from psoriatic tissue; is expressed in psoriatic tissue, e.g., psoriatic epidermis, at least 2, and preferably at least 5 or 10 times more abundantly than in normal tissue; is localized in the nucleus of the cell in psoriatic tissue.
In another aspect, the invention includes: a vector including a nucleic acid which encodes a psoriastatin type II polypeptide, e.g., a psoriastatin type II polypeptide; a host cell transfected with the vector; and a method of producing a recombinant psoriastatin type II-like polypeptide, e.g., a psoriastatin type II polypeptide; including culturing the cell, e.g., in a cell culture medium, and isolating the psoriastatin type II-like polypeptide, e.g., a psoriastatin type II polypeptide, e.g., from the cell or from the cell culture medium.
In another aspect, the invention features, a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleotide sequence shown in SEQ ID NO:3.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO:3, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a method of evaluating a compound for the ability to interact with, e.g., bind, or modulate, e.g., inhibit or promote, the activity of a psoriastatin polypeptide, e.g., a psoriastatin type I or a psoriastatin type II polypeptide. The method includes contacting the compound with the psoriastatin polypeptide, and evaluating ability of the compound to interact with or form a complex with the psoriastatin polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with psoriastatin polypeptides, e.g., psoriastatin type I or psoriastatin type II polypeptides. It can also be used to find natural or synthetic inhibitors of mutant or wild type psoriastatin polypeptides, e.g., psoriastatin type I or psoriastatin type II polypeptides. The compound can be a peptide or a non peptide molecule, e.g., a small molecule preferably 500 to 5,000 molecular weight, more preferably 500 to 1,000 molecular weight, having an aromatic scaffold, e.g., bis-amide phenols, decorated with various functional groups.
In brief, a two hybrid assay system (see e.g., Bartel et al. (1993) Cellular Interaction in Development: A practical Approach, D. A. Hartley, ed., Oxford University Press, Oxford, pp. 153-179) allows for detection of proteinxe2x80x94protein interactions in yeast cells. The known protein, e.g., a psoriastatin polypeptide, is often referred to as the xe2x80x9cbaitxe2x80x9d protein. The proteins tested for binding to the bait protein are often referred to as xe2x80x9cfishxe2x80x9d proteins. The xe2x80x9cbaitxe2x80x9d protein, e.g., a psoriastatin polypeptide, is fused to the GAL4 DNA binding domain. Potential xe2x80x9cfishxe2x80x9d proteins are fused to the GAL4 activating domain. If the xe2x80x9cbaitxe2x80x9d protein and a xe2x80x9cfishxe2x80x9d protein interact, the two GAL4 domains are brought into close proximity, thus rendering the host yeast cell capable of surviving a specific growth selection.
In another aspect, the invention features a method of identifying active fragments or analogs of a psoriastatin polypeptide, e.g., a psoriastatin type I or a psoriastatin type II polypeptide. The method includes first identifying a compound, e.g., cathepsin L, which interacts with a psoriastatin polypeptide and determining the ability of the compound to bind the fragment or analog. The two hybrid assay described above can be used to obtain fragment-binding compounds. These compounds can then be used as xe2x80x9cbaitxe2x80x9d to fish for and identify fragments of the psoriastatin polypeptide which interact, bind, or form a complex with these compounds.
In another aspect, the invention features a method of making a psoriastatin polypeptide, e.g., a psoriastatin type I or a psoriastatin type II polypeptide, having a non-wild amino acid sequence, e.g., an antagonist, agonist, or super agonist of a naturally occurring psoriastatin polypeptide, e.g., the psoriastatin type I or psoriastatin type II polypeptide. The method includes altering the sequence of a psoriastatin polypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4) by, for example, substitution or deletion of one or more residues of a conserved or non-conserved region, and testing the altered polypeptide for the desired activity, e.g., the ability to modulate, e.g., inhibit or promote, apoptosis or cell growth.
In another aspect, the invention features a method of making a psoriastatin polypeptide, e.g., a psoriastatin type I or a psoriastatin type II polypeptide, having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring psoriastatin polypeptide, e.g., the psoriastatin type I or psoriastatin type II polypeptide. The method includes altering the sequence of a psoriastatin polypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4) by, for example, substitution or deletion of one or more residues of a conserved or non-conserved region, and testing the altered polypeptide for the desired activity, e.g., the ability to modulate, e.g., inhibit or promote, apoptosis or cell growth.
In another aspect, the invention features a method of making a fragment or analog of a psoriastatin polypeptide e.g., a psoriastatin type I or a psoriastatin type II polypeptide, e.g., a psoriastatin polypeptide having at least one biological activity of a naturally occurring psoriastatin polypeptide. The method includes altering the sequence, e.g., by substitution or deletion of one or more residues, preferably which are conserved or non-conserved residues, of a psoriastatin polypeptide, and testing the altered polypeptide for the desired activity, e.g., the ability to modulate, e.g., inhibit or promote, apoptosis or cell growth.
In another aspect, the invention features a method of evaluating a compound for the ability to modulate psoriastatin polypeptide-, e.g., a psoriastatin type I or II polypeptide-, mediated regulation of cathepsin L. The method includes: contacting the compound with either or both of a psoriastatin polypeptide and cathepsin L, and determining the effect of the compound on the ability of psoriastatin to inhibit cathepsin L.
In another aspect, the invention features a method of evaluating a compound for the ability to modulate cathepsin L activity. The method includes: contacting the compound with either or both of psoriastatin polypeptide (e.g., psoriastatin type I or II polypeptide) and cathepsin L, and determining the effect of the compound on the activity of cathepsin L.
In another aspect, the invention features a method of treating a mammal, e.g., a human. The method includes administering to the mammal a therapeutically effective amount a substance which modulates an activity of psoriastatin type I or psoriastatin type II.
In another aspect, the invention features a method of treating a mammal e.g., a human, at risk for a disorder characterized by unwanted cell proliferation or less than wild type levels of apoptosis, e.g., cancer or psoriasis. The method includes administering to the mammal a treatment which modulates, e.g., inhibits, an activity of psoriastatin type I or psoriastatin type II, e.g., administering a therapeutically effective amount of a nucleic acid encoding an antagonist of a psoriastatin polypeptide, e.g., a psoriastatin type I or a psoriastatin type II polypeptide.
In another aspect, the invention features a method of treating a mammal, e.g., a human, at risk for a disorder characterized by unwanted cell proliferation or less than wild type levels of apoptosis, e.g., cancer or psoriasis. The method includes administering to the mammal a treatment which modulates, e.g., inhibits, an activity of psoriastatin type I or psoriastatin type II, e.g., administering a therapeutically effective amount of an antagonist of a psoriastatin polypeptide, e.g., an antagonist of a psoriastatin type I or a psoriastatin type II in polypeptide.
In preferred embodiments, the antagonist of psoriastatin polypeptide is: an antisense psoriastatin nucleic acid or an anti-psoriastatin antibody.
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a psoriastatin, e.g., a psoriastatin type I or type II, gene regulatory sequence. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid. In preferred embodiments the psoriastatin gene regulatory sequence is functionally linked to a heterologous gene, e.g., a reporter gene.
In another aspect, the invention features a human cell, e.g., a fibroblast or a dermal cell, e.g., a keratinocyte, transformed with a nucleic acid which encodes a psoriastatin polypeptide, e.g., a psoriastatin type I or a psoriastatin type II polypeptide.
In another aspect, the invention includes: an expression vector containing a nucleic acid encoding a psoriastatin polypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4), or an analog or fragment thereof; a cell transformed with an expression vector containing a nucleic acid encoding a psoriastatin polypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4), or an analog or fragment thereof; and a psoriastatin polypeptide made by culturing a cell transformed with an expression vector containing a nucleic acid encoding a psoriastatin polypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4), or an analog or fragment thereof.
In another aspect, the invention includes a transgenic animal, preferably a mammal, e.g., a mouse, rat, pig or goat, having a psoriastatin type I or psoriastatin type II transgene, e.g., a psoriastatin type I or psoriastatin type II gene having a deletion of all or a part of the wild type psoriastatin type I or psoriastatin type II gene. The transgenic animal can be heterozygous or homozygous for either or both of the transgenes.
Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed psoriastatin type I or II gene alleles or for use in drug screening. For example, the invention includes a method of evaluating the effect of the expression or misexpression of a psoriastatin type I or II gene on a parameter related to modulation, e.g., stimulation or inhibition, of cell proliferation or apoptosis. The method includes: providing a transgenic animal having a psoriastatin, e.g., psoriastatin type I and/or II, transgene, or which otherwise misexpresses a psoriastatin, e.g., psoriastatin type I and/or II, gene; contacting the animal with an agent; and evaluating the effect of the transgene on the parameter related to modulation, e.g., stimulation or inhibition, of cell proliferation or apoptosis.
In another aspect, the invention features a method of modulating cell proliferation or apoptosis, e.g., inhibiting or promoting cell proliferation or apoptosis, which includes modulating psoriastatin activity, e.g., psoriastatin type I or psoriastatin type II activity, e.g., by administering a compound which inhibits or promotes psoriastatin activity.
In another aspect, the invention features a method of inhibiting cell proliferation or promoting apoptosis which includes inhibiting psoriastatin activity. e.g., psoriastatin type I or psoriastatin type II activity, e.g., by administering an effective amount of a compound which inhibits psoriastatin activity.
In preferred embodiments, the psoriastatin activity is inhibited by: contacting, a cell, e.g., a dermal cell, e.g., a keratinocyte, with an antagonist of a psoriastatin polypeptide, e.g., psoriastatin type I and/or psoriastatin type II polypeptide, an antisense psoriastatin, or an anti-psoriastatin antibody.
In another aspect, the invention features a method of promoting cell proliferation or inhibiting apoptosis which includes promoting psoriastatin activity, e.g., psoriastatin type I or psoriastatin type II activity.
In preferred embodiments, the psoriastatin activity is promoted by: contacting a cell, e.g., a dermal cell, e.g., a keratinocyte, with a psoriastatin polypeptide, e.g., psoriastatin type I and/or psoriastatin type II polypeptide, or a psoriastatin polypeptide agonist
In another aspect, the invention features a method of modulating, e.g., promoting or inhibiting, cathepsin L activity including contacting a cell with an effective amount of a psonastatin, e.g., a psoriastatin type I and/or II polypeptide.
In another aspect, the invention features a method of treating a skin disorder. e.g., psoriasis, including administering to a subject a therapeutically effective amount of a compound which inhibits psoriastatin, e.g., a psoriastatin type I and/or II. activity.
In preferred embodiments, the compound which inhibits psoriastatin activity is any of: a peptide antagonist, an antibody, or an antisense molecule.
In preferred embodiments, the subject is a mammal, e.g., a rodent, e.g., a mouse or a rat, or a primate, e.g., a human.
A xe2x80x9cheterologous promoterxe2x80x9d, as used herein is a promoter which is not naturally associated with the psoriastatin, e.g., the psoriastatin type I or II, gene.
A xe2x80x9cpurified preparationxe2x80x9d or a xe2x80x9csubstantially pure preparationxe2x80x9d of a psoriastatin type I polypeptide, or a fragment or analog thereof, as used herein, means a psoriastatin type polypeptide, or a fragment or analog thereof, which is free of one or more other proteins including psoriastatin type II protein, lipids, and nucleic acids with which the psoriastatin type I polypeptide naturally occurs. Preferably, the polypeptide, or a fragment or analog thereof, is also separated from substances which are used to purify it, e.g., antibodies or gel matrix, such as polyacrylamide. Preferably, the polypeptide, or a fragment or analog thereof, constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 mg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A xe2x80x9cpurified preparationxe2x80x9d or a xe2x80x9csubstantially pure preparationxe2x80x9d of a psoriastatin type II polypeptide, or a fragment or analog thereof, as used herein, means a psoriastatin type II polypeptide, or a fragment or analog thereof, which is free from one or more other proteins including psoriastatin type I protein, lipids, and nucleic acids with which the psoriastatin type II polypeptide naturally occurs. Preferably, the polypeptide, or a fragment or analog thereof, is also separated from substances which are used to purify it. e.g., antibodies or gel matrix, such as polyacrylamide. Preferably, the polypeptide, or a fragment or analog thereof, constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 mg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A xe2x80x9cpurified preparation of cellsxe2x80x9d, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A xe2x80x9ctreatmentxe2x80x9d, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
A xe2x80x9csubstantially pure nucleic acidxe2x80x9d. e.g., a substantially pure DNA encoding a psoriastatin type I or II polypeptide, is a nucleic acid which is one or both of: not immediately contiguous with one or both of the coding sequences with which it is immediately contiguous (i.e. one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional psoriastatin type I or II sequences.
xe2x80x9cHomologousxe2x80x9d, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions comparedxc3x97100. For example, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
As used herein, the term xe2x80x9ctansgenexe2x80x9d means a nucleic acid sequence (encoding e.g., one or more psoriastatin type I and/or II polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence, such as the psoriastatin, e.g., the psoriastatin type I or II, gene, operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as neurons. The term also covers so-called xe2x80x9cleakyxe2x80x9d promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
A polypeptide has xe2x80x9cat least one biological activity of a psoriastatin type I polypeptidexe2x80x9d if it has one or more of the following properties: (1) the ability to react with an antibody, or antibody fragment, specific for (a) a wild type psoriastatin type I polypeptide, (b) a naturally-occurring mutant psoriastatin type I polypeptide, or (c) a fragment of either (a) or (b); (2) the ability to specifically inhibit cathepsin L activity; (3) the ability to promote cell proliferation or inhibit apoptosis; or (4) the ability to act as an antagonist or agonist of the activities recited in (1), (2) or (3).
A polypeptide has xe2x80x9cat least one biological activity of a psoriastatin type II polypeptidexe2x80x9d if it has one or more of the following properties: (1) the ability to react with an antibody, or antibody fragment, specific for (a) a wild type psoriastatin type II polypeptide, (b) a naturally-occurring mutant psoriastatin type II polypeptide, or (c) a fragment of either (a) or (b); (2) the ability to promote cell proliferation or inhibit apoptosis; (3) the ability to localize to the nucleus, or (4) the ability to act as an antagonist or agonist of the activities recited in (1), (2), or (3).
xe2x80x9cMisexpressionxe2x80x9d, as used herein, refers to a non-wild type pattern of psoriastatin type I or psoriastatin type II gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing, size, amino acid sequence, post-transitional modification, stability, or biological activity of the expressed psoriastatin type I or psoriastatin type II polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the psoriastatin type I or psoriastatin type II gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
As described herein, one aspect of the invention features a pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding a psoriastatin type I or psoriastatin type II polypeptide, and/or equivalents of such nucleic acids. The term xe2x80x9cnucleic acidxe2x80x9d, as used herein, can include fragments and equivalents. The term xe2x80x9cequivalentxe2x80x9d refers to nucleotide sequences encoding functionally equivalent polypeptides or functionally equivalent polypeptides which, for example, retain the ability to react with an antibody specific for a psoriastatin type I or psoriastatin type II polypeptide. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and will, therefore, include sequences that differ from the nucleotide sequence of psoriastatin shown in SEQ ID NO:1 or SEQ ID NO:3 due to the degeneracy of the genetic code.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., New York); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
The psoriatatin type I and psoriastatin type II genes and polypeptides of the present invention are useful for studying, diagnosing and/or treating diseases associated with unwanted cell proliferation or less than desired levels of apoptosis. e.g., psoriasis. The gene (or fragment thereof) can be used to prepare antisense constructs capable of inhibiting expression of a mutant or wild type psoriastatin type I or II gene encoding a polypeptide having an undesirable function. Alternatively, a psoriastatin type I or II polypeptide can be used to raise antibodies capable of detecting proteins or protein levels associated with psoriasis. An antagonist of psoriastatin type I or II can be administered to a patient afflicted with psoriasis to inhibit the activity of a wild type psoriastatin type I or II polypeptide. Furthermore, psoriastatin type I or II peptides, antibodies or nucleic acids, can be used to identify psoriatic tissue. Because psoriastatin peptides localize to the nucleus, they can be used to deliver compounds to the nucleus. Anti-psoriastatin antibodies can be used to identify the nucleus.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.